Sex-specific epigenetic development in the mouse hypothalamic arcuate nucleus pinpoints human genomic regions associated with body mass index.

Recent genome-wide association studies corroborate classical research on developmental programming indicating that obesity is primarily a neurodevelopmental disease strongly influenced by nutrition during critical ontogenic windows. Epigenetic mechanisms regulate neurodevelopment; however, little is known about their role in establishing and maintaining the brain's energy balance circuitry. We generated neuron and glia methylomes and transcriptomes from male and female mouse hypothalamic arcuate nucleus, a key site for energy balance regulation, at time points spanning the closure of an established critical window for developmental programming of obesity risk. We find that postnatal epigenetic maturation is markedly cell type and sex specific and occurs in genomic regions enriched for heritability of body mass index in humans. Our results offer a potential explanation for both the limited ontogenic windows for and sex differences in sensitivity to developmental programming of obesity and provide a rich resource for epigenetic analyses of developmental programming of energy balance.

The Role of the Gut Microbiota in the Effects of Early-Life Stress and Dietary Fatty Acids on Later-Life Central and Metabolic Outcomes in Mice.

Early-life stress (ELS) leads to increased vulnerability for mental and metabolic disorders. We have previously shown that a low dietary ω-6/ω-3 polyunsaturated fatty acid (PUFA) ratio protects against ELS-induced cognitive impairments. Due to the importance of the gut microbiota as a determinant of long-term health, we here study the impact of ELS and dietary PUFAs on the gut microbiota and how this relates to the previously described cognitive, metabolic, and fatty acid profiles. Male mice were exposed to ELS via the limited bedding and nesting paradigm (postnatal day (P)2 to P9 and to an early diet (P2 to P42) with an either high (15) or low (1) ω-6 linoleic acid to ω-3 alpha-linolenic acid ratio. 16S rRNA was sequenced and analyzed from fecal samples at P21, P42, and P180. Age impacted α- and ß-diversity. ELS and diet together predicted variance in microbiota composition and affected the relative abundance of bacterial groups at several taxonomic levels in the short and long term. For example, age increased the abundance of the phyla Bacteroidetes, while it decreased Actinobacteria and Verrucomicrobia; ELS reduced the genera RC9 gut group and Rikenella, and the low ω-6/ω-3 diet reduced the abundance of the Firmicutes Erysipelotrichia. At P42, species abundance correlated with body fat mass and circulating leptin (e.g., Bacteroidetes and Proteobacteria taxa) and fatty acid profiles (e.g., Firmicutes taxa). This study gives novel insights into the impact of age, ELS, and dietary PUFAs on microbiota composition, providing potential targets for noninvasive (nutritional) modulation of ELS-induced deficits. IMPORTANCE Early-life stress (ELS) leads to increased vulnerability to develop mental and metabolic disorders; however, the biological mechanisms leading to such programming are not fully clear. Increased attention has been given to the importance of the gut microbiota as a determinant of long-term health and as a potential target for noninvasive nutritional strategies to protect against the negative impact of ELS. Here, we give novel insights into the complex interaction between ELS, early dietary ω-3 availability, and the gut microbiota across ages and provide new potential targets for (nutritional) modulation of the long-term effects of the early-life environment via the microbiota.

Modulation of the Hypothalamic Nutrient Sensing Pathways by Sex and Early-Life Stress.

There are sex differences in metabolic disease risk, and early-life stress (ES) increases the risk to develop such diseases, potentially in a sex-specific manner. It remains to be understood, however, how sex and ES affect such metabolic vulnerability. The hypothalamus regulates food intake and energy expenditure by sensing the organism's energy state via metabolic hormones (leptin, insulin, ghrelin) and nutrients (glucose, fatty acids). Here, we investigated if and how sex and ES alter hypothalamic nutrient sensing short and long-term. ES was induced in mice by limiting the bedding and nesting material from postnatal day (P)2-P9, and the expression of genes critical for hypothalamic nutrient sensing were studied in male and female offspring, both at P9 and in adulthood (P180). At P9, we observed a sex difference in both Ppargc1a and Lepr expression, while the latter was also increased in ES-exposed animals relative to controls. In adulthood, we found sex differences in Acacb, Agrp, and Npy expression, whereas ES did not affect the expression of genes involved in hypothalamic nutrient sensing. Thus, we observe a pervasive sex difference in nutrient sensing pathways and a targeted modulation of this pathway by ES early in life. Future research is needed to address if the modulation of these pathways by sex and ES is involved in the differential vulnerability to metabolic diseases.

Characterization of bacterial cellulose nanocrystals: Effect of acid treatments and neutralization.

In this study, bacterial cellulose nanocrystals (BCNCs) were obtained from bacterial cellulose nanofibers (BCNFs) by controlled hydrolysis of sulfuric and hydrochloric acids. The influence of hydrolysis temperature and acid type with the addition of the post-treatment step was studied. The obtained BCNCs were analyzed based on the structural characterization and the properties of the nanocrystals. The BCNCs crystallinity increased, and the size of nanocrystals decreased with increasing 10 °C hydrolysis temperature for both acid hydrolysis conditions. Hydrolysis conditions with neutralization post-treatment did not alter the thermal stability of nanocrystals, and BCNCs had high thermal stability like raw BCNFs. Elemental analysis results indicated that sulfur content (S %) was very low for sulfuric acid hydrolyzed samples, and X-ray results did not show any sulfate salt peaks. Thermal stable BCNCs with high crystallinity were successfully produced to meet the process requirements in various applications, especially in the food industry.

Development of sodium chlorite and glucono delta-lactone incorporated PLA film for microbial inactivation on fresh tomato.

Chlorine dioxide (ClO2) is an effective disinfectant used in the sanitization of fresh produce. Glucono delta-lactone (GDL), widely used as an acidifier during food processing, can be partially hydrolyzed to become a weak acid-gluconic acid under chemical equilibrium upon dissolution in water. This study focused on the development of a novel polylactic acid (PLA) film which incorporated with sodium chlorite (NaClO2) and GDL for ClO2(g) generation. The effects of PLA amount, NaClO2 + GDL/PLA ratio, NaClO2/GDL ratio, temperature and relative humidity on the release profiles of ClO2(g) were elucidated. The storage test indicated that film efficacy was well maintained after 4 weeks of storage under ambient conditions. The microbial inactivation results revealed that ClO2(g) generated from the films reduced populations of surface-inoculated Salmonella and Escherichia coli O157:H7 from ca. 5 log CFU/tomato to undetectable level (<1 log CFU/tomato) within 2 and 4 h respectively and the complete elimination in populations of both bacterial species was maintained throughout the 14-day storage period at both 10 and 22 °C. The sensory properties of treated tomatoes were evaluated and exhibited no significant difference (p > 0.05) compared to controls except for appearance on day 14 under 22 °C storage.

A Review on Flavonoid Apigenin: Dietary Intake, ADME, Antimicrobial Effects, and Interactions with Human Gut Microbiota.

Apigenin is a flavonoid of low toxicity and multiple beneficial bioactivities. Published reviews all focused on the findings using eukaryotic cells, animal models, or epidemiological studies covering the pharmacokinetics, cancer chemoprevention, and drug interactions of apigenin; however, no review is available on the antimicrobial effects of apigenin. Research proves that dietary apigenin passes through the upper gastrointestinal tract and reaches the colon after consumption. For that reason, it is worthwhile to study the potential interactions between apigenin and human gut microbiota. This review summarizes studies on antimicrobial effects of apigenin as well as what has been reported on apigenin and human gut microbiota. Various levels of effectiveness have been reported on apigenin's antibacterial, antifungal, and antiparasitic capability. It has been shown that apigenin or its glycosides are degraded into smaller metabolites by certain gut bacteria which can regulate the human body after absorption. How apigenin contributes to the structural and functional changes in human gut microbiota as well as the bioactivities of apigenin bacterial metabolites are worth further investigation.

Increasing availability of ω-3 fatty acid in the early-life diet prevents the early-life stress-induced cognitive impairments without affecting metabolic alterations.

Exposure to early-life stress (ES) is associated with cognitive and metabolic deficits in adulthood. The role of early nutrition in programming these long-term effects is largely unknown. We focused on essential ω-3 and ω-6 long-chain polyunsaturated fatty acids (LCPUFA) and investigated whether ES affects central and peripheral FA profiles, as well as if and how an early diet with increased availability of ω-3 LCPUFA ( via lowering ω-6/ω-3 ratio) protects against ES-induced impairments. ES exposure [limited nesting and bedding paradigm from postnatal day (P)2 to P9] altered central and peripheral FA profiles in mice. An early diet with low ω-6/ω-3 ratio from P2 to P42 notably prevented the ES-induced cognitive impairments, and the alterations in hippocampal newborn cell survival and in CD68+ microglia, without affecting the ES-induced metabolic alterations. Other markers for hippocampal plasticity, apoptosis, and maternal care were unaffected by ES or diet. Our findings highlight the importance of early dietary lipid quality for later cognition in ES-exposed populations.-Yam, K.-Y., Schipper, L., Reemst, K., Ruigrok, S. R., Abbink, M. R., Hoeijmakers, L., Naninck, E. F. G., Zarekiani, P., Oosting, A., Van der Beek, E. M., Lucassen, P. J., Korosi, A. Increasing availability of ω-3 fatty acid in the early-life diet prevents the early-life stress-induced cognitive impairments without affecting metabolic alterations.

Critical review of controlled release packaging to improve food safety and quality.

Controlled release packaging (CRP) is an innovative technology that uses the package to release active compounds in a controlled manner to improve safety and quality for a wide range of food products during storage. This paper provides a critical review of the uniqueness, design considerations, and research gaps of CRP, with a focus on the kinetics and mechanism of active compounds releasing from the package. Literature data and practical examples are presented to illustrate how CRP controls what active compounds to release, when and how to release, how much and how fast to release, in order to improve food safety and quality.

Hyperleptinemia in Neonatally Overfed Female Rats Does Not Dysregulate Feeding Circuitry.

Neonatal overfeeding during the first weeks of life in male rats is associated with a disruption in the peripheral and central leptin systems. Neonatally overfed male rats have increased circulating leptin in the first 2 weeks of life, which corresponds to an increase in body weight compared to normally fed counterparts. These effects are associated with a short-term disruption in the connectivity of neuropeptide Y (NPY), agouti-related peptide (AgRP), and pro-opiomelanocortin (POMC) neurons within the regions of the hypothalamus responsible for control of energy balance and food intake. Female rats that are overfed during the first weeks of their life experience similar changes in circulating leptin levels as well as in their body weight. However, it has not yet been studied whether these metabolic changes are associated with the same central effects as observed in males. Here, we hypothesized that hyperleptinemia associated with neonatal overfeeding would lead to changes in central feeding circuitry in females as it does in males. We assessed hypothalamic NPY, AgRP, and POMC gene expression and immunoreactivity at 7, 12, or 14 days of age, as well as neuronal activation in response to exogenous leptin in neonatally overfed and control female rats. Neonatally overfed female rats were hyperleptinemic and were heavier than controls. However, these metabolic changes were not mirrored centrally by changes in hypothalamic NPY, AGRP, and POMC fiber density. These findings are suggestive of sex differences in the effects of neonatal overfeeding and of differences in the ability of the female and male central systems to respond to changes in the early life nutritional environment.

Apigenin Impacts the Growth of the Gut Microbiota and Alters the Gene Expression of Enterococcus.

Apigenin is a major dietary flavonoid with many bioactivities, widely distributed in plants. Apigenin reaches the colon region intact and interacts there with the human gut microbiota, however there is little research on how apigenin affects the gut bacteria. This study investigated the effect of pure apigenin on human gut bacteria, at both the single strain and community levels. The effect of apigenin on the single gut bacteria strains Bacteroides galacturonicus, Bifidobacterium catenulatum, Lactobacillus rhamnosus GG, and Enterococcus caccae, was examined by measuring their anaerobic growth profiles. The effect of apigenin on a gut microbiota community was studied by culturing a fecal inoculum under in vitro conditions simulating the human ascending colon. 16S rRNA gene sequencing and GC-MS analysis quantified changes in the community structure. Single molecule RNA sequencing was used to reveal the response of Enterococcus caccae to apigenin. Enterococcus caccae was effectively inhibited by apigenin when cultured alone, however, the genus Enterococcus was enhanced when tested in a community setting. Single molecule RNA sequencing found that Enterococcus caccae responded to apigenin by up-regulating genes involved in DNA repair, stress response, cell wall synthesis, and protein folding. Taken together, these results demonstrate that apigenin affects both the growth and gene expression of Enterococcus caccae.

Pectic oligosaccharide structure-function relationships: Prebiotics, inhibitors of Escherichia coli O157:H7 adhesion and reduction of Shiga toxin cytotoxicity in HT29 cells.

Shiga toxin (Stx)-producing, food-contaminating Escherichia coli (STEC) is a major health concern. Plant-derived pectin and pectic-oligosaccharides (POS) have been considered as prebiotics and for the protection of humans from Stx. Of five structurally different citrus pectic samples, POS1, POS2 and modified citrus pectin 1 (MCP1) were bifidogenic with similar fermentabilities in human faecal cultures and arabinose-rich POS2 had the greatest prebiotic potential. Pectic oligosaccharides also enhanced lactobacilli growth during mixed batch faecal fermentation. We demonstrated that all pectic substrates were anti-adhesive for E. coli O157:H7 binding to human HT29 cells. Lower molecular weight and deesterification enhanced the anti-adhesive activity. We showed that all pectic samples reduced Stx2 cytotoxicity in HT29 cells, as measured by the reduction of human rRNA depurination detected by our novel TaqMan-based RT-qPCR assay, with POS1 performing the best. POS1 competes with Stx2 binding to the Gb3 receptor based on ELISA results, underlining the POS anti-STEC properties.

Innovative application of metal-organic frameworks for encapsulation and controlled release of allyl isothiocyanate.

This research investigated the technical feasibility of metal-organic frameworks (MOFs) as novel delivery systems for encapsulation and controlled release of volatile allyl isothiocyanate (AITC) molecules. We hypothesized that water vapor molecules could act as an external stimulus to trigger the release of AITC molecules encapsulated in MOFs. To test this hypothesis, three MOFs-HKUST-1, MOF-74(Zn), and RPM6-Zn-were selected based on their structural properties and AITC molecular characteristics. Results from adsorption-desorption and GC headspace analyses showed that these MOFs could encapsulate and retain AITC molecules within their pores under low (30-35%) relative humidity (RH) conditions. In contrast, the release of AITC molecules from all these MOFs was triggered under high RH (95-100%) conditions. These findings along with results from SEM, TEM, and XRPD studies support our hypothesis that water vapors could trigger the AITC release from these MOFs, indicating that development of the AITC-MOFs delivering system is technically feasible.

Early micronutrient supplementation protects against early stress-induced cognitive impairments.

Early-life stress (ES) impairs cognition later in life. Because ES prevention is problematic, intervention is needed, yet the mechanisms that underlie ES remain largely unknown. So far, the role of early nutrition in brain programming has been largely ignored. Here, we demonstrate that essential 1-carbon metabolism-associated micronutrients (1-CMAMs; i.e., methionine and B vitamins) early in life are crucial in programming later cognition by ES. ES was induced in male C57Bl/6 mice from postnatal d (P)2-9. 1-CMAM levels were measured centrally and peripherally by using liquid chromatography-mass spectroscopy. Next, we supplemented the maternal diet with 1-CMAM only during the ES period and studied cognitive, neuroendocrine, neurogenic, transcriptional, and epigenetic changes in adult offspring. We demonstrate that ES specifically reduces methionine in offspring plasma and brain. Of note, dietary 1-CMAM enrichment during P2-9 restored methionine levels and rescued ES-induced adult cognitive impairments. Beneficial effects of this early dietary enrichment were associated with prevention of the ES-induced rise in corticosterone and adrenal gland hypertrophy did not involve changes in maternal care, hippocampal volume, neurogenesis, or global/Nr3c1-specific DNA methylation. In summary, nutrition is important in brain programming by ES. A short, early supplementation with essential micronutrients can already prevent lasting effects of ES. This concept opens new avenues for nutritional intervention.-Naninck, E. F. G., Oosterink, J. E., Yam, K.-Y., de Vries, L. P., Schierbeek, H., van Goudoever, J. B., Verkaik-Schakel, R.-N., Plantinga, J. A., Plosch, T., Lucassen, P. J., Korosi, A. Early micronutrient supplementation protects against early stress-induced cognitive impairments.

Effects of Ultraviolet (UV) on Degradation of Irgafos 168 and Migration of Its Degradation Products from Polypropylene Films.

The effects of ultraviolet (UV) irradiation on the degradation of Irgafos 168 and the migration of its two degradation products, 2,4-di-tert-butylphenol and tris(2,4-di-tert-butylphenyl)phosphate, from polypropylene (PP) were investigated. A blown film machine was used to extrude PP films containing Irgafos 168, the films were stored in the dark for 45 days, two UV treatments and sunlight exposure were applied to the films, and GC-MS was used for degradation and migration studies. Extrusion, storage, UV treatments, and sunlight exposure significantly affected concentrations of Irgafos 168 and the degradation products. 2,4-Di-tert-butylphenol was the major degradation product produced by UV irradiation, but tris(2,4-di-tert-butylphenyl)phosphate was the major degradation product produced by extrusion, storage, and sunlight exposure. The degradation products have no or little health risk, because migration study and threshold of toxicological concern (TTC) analysis show that experimental maximum migration of 2,4-di-tert-butylphenol and tris(2,4-di-tert-butylphenyl)phosphate are only 2 and 53% of the theoretical maximum migration amounts, respectively.

The moisture-triggered controlled release of a natural food preservative from a microporous metal-organic framework.

In this work we demonstrate that allyl isothiocyanate (AITC), a common food flavoring agent and food preservative, can be effectively captured by and released in a controlled manner from a microporous metal-organic framework (MOF). The extent of AITC-MOF interactions is quantitatively measured by orbital overlap population calculations. Controlled release experiments show that loaded AITC can be released by applying higher relative humidity. Further analysis reveals that the underlying mechanism of the controlled release is associated with the transformation of the MOF from a porous to a nonporous structure at high humidity. This study represents the first example of making use of MOF porosity in food preservation.

Early-life adversity programs emotional functions and the neuroendocrine stress system: the contribution of nutrition, metabolic hormones and epigenetic mechanisms.

Clinical and pre-clinical studies have shown that early-life adversities, such as abuse or neglect, can increase the vulnerability to develop psychopathologies and cognitive decline later in life. Remarkably, the lasting consequences of stress during this sensitive period on the hypothalamic-pituitary-adrenal axis and emotional function closely resemble the long-term effects of early malnutrition and suggest a possible common pathway mediating these effects. During early-life, brain development is affected by both exogenous factors, like nutrition and maternal care as well as by endogenous modulators including stress hormones. These elements, while mostly considered for their independent actions, clearly do not act alone but rather in a synergistic manner. In order to better understand how the programming by early-life stress takes place, it is important to gain further insight into the exact interplay of these key elements, the possible common pathways as well as the underlying molecular mechanisms that mediate their effects. We here review evidence that exposure to both early-life stress and early-life under-/malnutrition similarly lead to life-long alterations on the neuroendocrine stress system and modify emotional functions. We further discuss how the different key elements of the early-life environment interact and affect one another and next suggest a possible role for the early-life adversity induced alterations in metabolic hormones and nutrient availability in shaping later stress responses and emotional function throughout life, possibly via epigenetic mechanisms. Such knowledge will help to develop intervention strategies, which gives the advantage of viewing the synergistic action of a more complete set of changes induced by early-life adversity.

Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells.

Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the residue remaining following commercial juice extraction that included pectinase maceration of the pulp. A novel xyloglucan was detected through tandem mass spectrometry analysis of an ion at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp (1→6) ß-D-Glcp sequence, proving the S-type cranberry xyloglucan structure. Cranberry xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24 human bladder epithelial cells and that of E. coli O157:H7 to HT29 human colonic epithelial cells. SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli anti-adhesion activity and high affinity for type 1 fimbriae.

Modeling the impact of vapor thymol concentration, temperature, and modified atmosphere condition on growth behavior of Salmonella on raw shrimp.

Salmonella is a microorganism of concern on a global basis for raw shrimp. This research modeled the impact of vapor thymol concentration (0, 0.8, and 1.6 mg/liter), storage temperature (8, 12, and 16°C), and modified atmosphere condition (0.04 as in the natural atmosphere and 59.5% CO2) against the growth behavior of a Salmonella cocktail (six strains) on raw shrimp. Lag time (hour) and maximum growth rate (log CFU per gram per hour), chosen as two growth indicators, were obtained through DMFit software and then developed into polynomial as well as nonlinear modified secondary models (dimensional and/or dimensionless), consisting of two or even three impact factors in the equations. The models were validated, and results showed that the predictive values from both models demonstrated good matches to the observed experimental values, yet the prediction based on lag time was more accurate than maximum growth rate. The information will provide the food industry with insight into the potential safety risk of Salmonella growth on raw shrimp under stressed conditions.

Evaluation of chlorine dioxide gas treatment to inactivate Salmonella enterica on mungbean sprouts.

Although freshly sprouted beans and grains are considered to be a source of nutrients, they have been associated with foodborne outbreaks. Sprouts provide good matrices for microbial localization and growth due to optimal conditions of temperature and humidity while sprouting. Also, the lack of a kill step postsprouting is a major safety concern. The objective of this work was to evaluate the effectiveness of chlorine dioxide gas treatment to reduce Salmonella on artificially inoculated mungbean sprouts. The effectiveness of gaseous chlorine dioxide (0.5 mg/liter of air) with or without tumbling (mechanical mixing) was compared with an aqueous chlorine (200 ppm) wash treatment. Tumbling the inoculated sprouts during the chlorine dioxide gas application for 15, 30, and 60 min reduced Salmonella populations by 3.0, 4.0, and 5.5 log CFU/g, respectively, as compared with 3.0, 3.0, and 4.0 log CFU/g reductions obtained without tumbling, respectively. A 2.0 log CFU/g reduction in Salmonella was achieved with an aqueous chlorine wash. The difference in microbial reduction between chlorine dioxide gas versus aqueous chlorine wash points to the important role of surface topography, pore structure, bacterial attachment, and/or biofilm formation on sprouts. These data suggested that chlorine dioxide gas was capable of penetrating and inactivating cells that are attached to inaccessible sites and/or are within biofilms on the sprout surface as compared with an aqueous chlorine wash. Consequently, scanning electron microscopy imaging indicated that chlorine dioxide gas treatment was capable of penetrating and inactivating cells attached to inaccessible sites and within biofilms on the sprout surfaces.

Antimicrobial effects of vapor phase thymol, modified atmosphere, and their combination against Salmonella spp. on raw shrimp.

Salmonella contamination on raw shrimp is a big food safety concern in the United States currently. This research evaluated the inhibition effects of vapor phase thymol, modified atmosphere (MA), and their combination against Salmonella spp. on raw shrimp. Growth profiles of a Salmonella spp. cocktail (6 strains), inoculated onto the surface of raw shrimp, treated with vapor phase thymol at 3 levels (0, 0.8, and 1.6 mg/L), or MA (59.5% CO2 + 39.5% N2 + 1% O2 ), both alone and in combination, at 3 temperatures (8, 12, and 16 ºC), were determined. Lag time and maximum growth rate of Salmonella spp. under each treatment were obtained using Baranyi and Roberts models. Results indicated that both vapor phase thymol and MA treatments alone inhibited the growth potential of Salmonella spp. effectively, extending the lag time by 10% to 100% and reducing the maximum growth rate by 14% to 71% compared with controlled samples at experimental temperatures (8, 12, and 16 ºC). Combination treatments of vapor phase thymol and MA exhibited greater inhibition effectiveness than each individual treatment and a synergistic antimicrobial effectiveness could be observed on the lag time extension. To the maximum, at 12 ºC, lag time of Salmonella spp. was extended 59.6% more by the combination treatment of 0.8 mg/L thymol + MA (36.97 h) than those effects combined from 0.8 mg/L thymol treatment and MA treatment alone (23.16 h in total). This combination strategy could be potentially utilized for Salmonella inhibition during the long distance and temperature-abused raw shrimp import process.